This project is a study of the structure, function, and splicing mechanisms of the group II and group III introns and twintrons ("introns-within-introns") of the plastid genomes of Euglena gracilis and related protists. These organisms are the richest known source of group II introns, and have the only known group II and group III twintrons. The specific aims include: (1) Characterization of novel group II and group III introns and twintrons in species related to Euglena gracilis as a means to explore (i) possible "founder" events of intron invasion, (ii) whether group II and group III intorns shared a common conserved structural motifs required for RNA splicing; (2) An investigation of the role of the group III intron spicing, and (3) Analysis of cis-acting domains of group II and group III intorns required for in vivo splicing in transgenic I. gracilis chloroplasts. This aim will be expanded to include site-directed mutagenesis of intron maturases if sufficient technical progress in plastid transformation is achieved. This work is addressed at understanding fundamental questions about the origins and evolution of introns and twintrons. A working hypothesis is that group II and group III introns, as well as nuclear pre-mRNA spliceosomal intorns, may have all shared a common evolutionary ancestor. Therefore, studies on unusual variants among the smallest group II and group III introns should be relevant to understanding the RNA structures at the heart of all splicing reactions.